Bedside to Bench: Role of Muscarinic Receptor Activation in Ultrarapid Growth of Colorectal Cancer in a Patient With Pheochromocytoma  Erik C. von Rosenvinge, MD, Kunrong Cheng, PhD, Cinthia B. Drachenberg, MD, Carol B. Fowler, PhD, David L. Evers, PhD, Guofeng Xie, MD, PhD, Jean-Pierre Raufman, MD  Mayo Clinic Proceedings  Volume 88, Issue 11, Pages 1340-1346 (November 2013) DOI: 10.1016/j.mayocp.2013.06.023 Copyright © 2013 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 1 Endoscopic findings in a patient with long-standing unresectable pheochromocytoma who experienced rapid development of invasive rectal adenocarcinoma after removal of a rectal adenoma. A, Rectal polyp before initial polypectomy. B, Polypectomy site 6 months later when additional polypectomy revealed residual adenoma. C, Appearance of polypectomy scar 3 months after second procedure. Multiple biopsies obtained at this time showed no adenomatous tissue. D, Rectal adenocarcinoma found 28 months after the unremarkable sigmoidoscopy with biopsy (white arrows outline the proximal tumor margin). Mayo Clinic Proceedings 2013 88, 1340-1346DOI: (10.1016/j.mayocp.2013.06.023) Copyright © 2013 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 2 Photomicrographs of specimens from the patient’s pheochromocytoma. A, Histopathologic evaluation of the pheochromocytoma revealed connective tissue with clusters and isolated pleomorphic tumor cells with abundant and granular cytoplasm (hematoxylin-eosin, original magnification ×200). B, Immunohistochemical staining for choline acetyltransferase (ChAT) produced strong cytoplasmic staining of most tumor cells in this field (anti-ChAT stain, original magnification ×200). C and D, Two of 5 additional archival specimens from other patients stained positively for ChAT (anti-ChAT stain, original magnification ×200). Mayo Clinic Proceedings 2013 88, 1340-1346DOI: (10.1016/j.mayocp.2013.06.023) Copyright © 2013 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 3 Muscarinic receptor subtype 3 (M3R) gene and protein expression. A, Comparison of muscarinic receptor subtype gene expression in the patient’s colorectal tumor compared with adjacent normal colon tissue. The dashed line represents equivalence. Results were obtained by real-time polymerase chain reaction using the 2-ΔΔCt comparative method with GAPDH as the relative control. B, Immunohistochemical staining for M3R (see “Methods” section) revealed weak staining in normal colon mucosa (1), whereas robust staining was observed in a fragment of the tubular adenoma removed by endoscopic biopsy before progression to cancer (2). Staining for M3R in an endoscopic biopsy specimen from the moderately well-differentiated colorectal neoplasm (3) was less intense than that observed in the polyp (2) but substantially greater than that observed in normal colon (1) (anti-M3R stain, original magnification ×400). Mayo Clinic Proceedings 2013 88, 1340-1346DOI: (10.1016/j.mayocp.2013.06.023) Copyright © 2013 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 4 Results of conditioned media experiments. Media exposed to PC-12 pheochromocytoma cells (“conditioned media”) stimulated proliferation of H508 human colon cancer cells. H508 cell proliferation was determined by absorbance at 490 nm as described in the “Methods” section. The proliferative effects of conditioned media were significantly attenuated in the presence of 5 μM of atropine (At). Data represent means and SEs from 3 independent experiments. Mayo Clinic Proceedings 2013 88, 1340-1346DOI: (10.1016/j.mayocp.2013.06.023) Copyright © 2013 Mayo Foundation for Medical Education and Research Terms and Conditions